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The BuShen JiangZhi (BSJZ) recipe is a Chinese medicine compound with the effect of tonifying the kidney, replenishing essence, and lowering blood fat to unblock vessels. The purpose of this study is to explore whether the mechanism of BSJZ for effective intervention in the treatment of AS is related to mmu_circRNA_22187 and aminopeptidase N (Anpep). ApoE-/- mice were induced by a high-fat diet to replicate the AS model. 24 ApoE-/- mice were randomly divided into model group (group M), BSJZ group (group BS), and 12 C57BL/6 mice of the same genetic background and same weeks of age as the normal control group (group C). Mice in the BS group were given an aqueous solution of BSJZ by gavage, while mice in groups C and M were given the same volume of distilled water. HE and Oil Red O staining were used to detect the pathomorphology and lipid accumulation of mouse aortic sinus. Arraystar version 2.0 mouse circRNA chip was used to scan with Agilent Scanner G2505C, and the differential circRNAs expression profile of mice aorta was obtained. Scatter plot, volcano plot, and cluster map, respectively, visualized the differentially expressed circRNAs, as well as the types of circRNAs and the chromosomes’ distribution, screened and compared the differentially expressed circRNAs intersection between groups by Venny software, and then combined ceRNA bioinformatics analysis to construct a ceRNA network. The results showed that BSJZ could significantly reduce the area of AS plaque and lipid accumulation in the aortic sinus of ApoE-/- mice induced by a high-fat diet. The bioinformatics analysis showed that mmu_circRNA_22187 may be a key circRNA of BSJZ intervention in the treatment of AS. Compared with group C, the expressions of Anpep mRNA and protein were upregulated in group M. After the intervention of BSJZ, the expressions of Anpep mRNA and protein were downregulated. Therefore, BSJZ could effectively treat AS which might be related to the regulation of mmu_circRNA_22187 and Anpep.

A high-fat diet and sedentary lifestyle could accelerate aging and hypothalamic inflammation. In order to explore the regulatory mechanisms of lifestyle in the hypothalamus, swimming exercise and diet control were applied in the high-fat diet ApoE-/- mice in our study. 20-week-old ApoE-/- mice fed with 12-week high-fat diet were treated by high-fat diet, diet control and swimming exercise. The results showed that hypothalamic inflammation, glial cells activation and cognition decline were induced by high-fat diet. Compared with the diet control, hypothalamic inflammation, glial cells activation and learning and memory impairment were effectively alleviated by swimming exercise plus diet control, which was related to the increasing expression of SIRT1, inhibiting the expression of NF-κB and raising secretion of GnRH in the hypothalamus. These findings supported the hypothesis that hypothalamic inflammation was susceptible to exercise and diet, which was strongly associated with SIRT1-NF-κB-GnRH expression in the hypothalamus.

High-fat diet (HFD) is the primary cause of metabolic syndrome associated chronic kidney disease. This study aimed to investigate the pathogenesis of HFD-induced kidney injury. ApoE−/− mice were fed with HFD and kidney damage was examined. In addition, HK-2 human renal proximal tubular epithelial cells were treated with fructose and receptor of advanced glycation end products (RAGE) siRNA. The results showed that HFD increased body weight, blood glucose and insulin resistance in ApoE−/− mice. The kidney damage was associated with increased oxidative stress and strong staining of RAGE and NF-κB in kidney tissues, as well as high serum levels of TNF-α, IL-1β and IL-6. Western-blot analysis showed that HFD increased the levels of RAGE, p-IκBα, p-NF-κB, bax, caspase-3 and caspase-9 but decreased the levels of Bcl-2 in kidney tissues. In HK-2 cells, fructose promoted the secretion of TNF-α, IL-1β and IL-6 and increased the levels of RAGE, p-IκBα, p-NF-κB, bax, caspase-3 and caspase-9, but decreased the levels of Bcl-2. Moreover, RAGE siRNA could attenuate increased levels of p-IκBα, p-NF-κB, bax, caspase-3 and caspase-9 while restore decreased levels of Bcl-2 in fructose-treated HK-2 cells. In conclusion, HFD causes kidney injury by promoting oxidative stress, inflammation and apoptosis possibly through the activation of RAGE/NF-κB pathway.

It is increasingly aware that gut microbiota is closely associated with atherosclerosis. However, which and how specific gut bacteria regulate the progression of atherosclerosis is still poorly understood. In this study, modified linear discriminant analysis was performed in comparing the gut microbiota structures of atherosclerotic and non-atherosclerotic mice, and Desulfovibrio desulfuricans (D. desulfuricans) was found to be associated with atherosclerosis. D. desulfuricans-treated Apoe−/− mice showed significantly aggravated atherosclerosis. The proatherogenic effect of D. desulfuricans was attributed to its ability to increase intestinal permeability and subsequent raise in the transit of lipopolysaccharide (LPS) from the intestine to the bloodstream. Excessive LPS in the blood can elicit local and systemic inflammation and activate Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling of endothelial cells. TAK-242, a specific inhibitor of TLR4, can ameliorate the development of D. desulfuricans-induced atherosclerosis by blocking the LPS-induced activation of TLR4/NF-κB signaling.

Yifan Cai,1 Qiutang Zeng,1 Yuzhou Liu,2 Ruirui Zhu,1 Kunwu Yu,1 Wenbin Xu,1 Yue Wang,1 Yan Ding,1 Jian Yu,1 Chengliang Pan,1 Yudong Peng,1 Yi Mao,1 Peng Cheng,1 Lun Huang,1 Xiaobo Mao,1 Yucheng Zhong1 1Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, People’s Republic of China; 2Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, People’s Republic of ChinaCorrespondence: Yucheng ZhongLaboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jie-Fang Avenue, Wuhan, 430022, People’s Republic of ChinaTel +86-27-85726423Fax +86-27-85726425Email zyc811029@126.comPurpose: This study aims to clarify the specific mechanism by which GARP affects the atherosclerotic plaques in ApoE−/- mice and the effect of GARP-tDC on atherosclerosis.Methods: The mice were randomly divided into three groups: the control group, the GARP-overexpressed group and the GARP-inhibited group. After 12 weeks, all the mice were euthanized, and the specimens were collected. In vitro, experiments were conducted to observe the effect of GARP on DC phenotype and the changes of the proportion of CD4+CD25+Foxp3+ Treg cells when GARP-tDCs were co-cultured with CD4+ T cells. Furthermore, adoptive transmission of GARP-tDCs was used to observe the effect on atherosclerotic plaque in mice.Results: The GARP-overexpressed group enhanced the biological activity of Foxp3+ CD4+CD25+ Tregs and resulted in increased expression of LAP in T cells. In addition, the GARP-overexpressed group significantly suppressed the function of Th1 and Th17, and decreased the secretion of INF-γ and IL-17A. Thus, GARP had a protective effect on atherosclerosis. In vitro, we found that GARP-tDC had a tolerance-inducing phenotype, and GARP-tDC also had the ability to induce tolerance when co-cultured with CD4+ T cells. More importantly, adoptive transmission of GARP-tDCs reduced the size of atherosclerotic plaques.Conclusion: GARP and the GARP-tDC play protective roles in atherosclerosis. The protective effect of GARP on atherosclerosis is achieved by increasing CD4+CD25+Foxp3+ Treg cells and inhibiting the production of IFN-γ and IL-17A.Keywords: GARP, Foxp3, GARP-tDC, atherosclerosis, LAP

Lactoferrin (LF) is a multifunctional glycoprotein and has beneficial effects on the regulation of lipid metabolism. However, whether LF supplementation alleviates the development of ather...

The aim of this study is to detect the dynamic expression of interleukin-23 (IL-23) in ApoE-/- mice at different ages and to further examine the effects of anti-IL-23 therapy on atherosclerosis development. The levels of IL-23 in the sera, aortas, and lymph nodes of ApoE-/- mice were significantly increased compared with those of age-matched controls at 8, 12, 16, 20, and 24 weeks of age. Then, 12-week-old ApoE-/- mice were intraperitoneally injected with anti-IL-23p19 neutralizing antibodies, isotype controls, and phosphate-buffered saline for 8 weeks. The proinflammatory and anti-inflammatory mediators in atherosclerotic aortas, plaque areas, plaque necrotic cores, and the contents of major inflammatory cells in plaques were subsequently determined. The results showed that anti-IL-23p19 treatment significantly decreased the expression of IL-17A, IL-6, and TNF-α in the aortas of ApoE-/- mice, but had no obvious effect on the plaque area, plaque necrotic core, or content of major inflammatory cells in atherosclerotic plaques. Although anti-IL-23p19 therapy reduces the expression of several proinflammatory cytokines, it does not significantly suppress the progression of atherosclerosis in ApoE-/- mice.

As one of the most nutrient-rich marine species, Thelenota ananas has various effects, but its antiatherosclerosis activity has yet to be plumbed in depths. In this work, T. ananas saponin extracts were used to assess the effect on atherosclerosis development in the apoE−/− mice. In detail, the saponin treated groups clearly showed an inhibitory effect on the size of aortic atheromatous plaque, and the levels of serum lipid and inflammatory cytokines compared with the atherosclerosis model mice. Interestingly, the saponin extracts decreased the lipid profiles and oxidation in the visceral fat, and markedly altered the expressions of key proteins related to lipid synthesis and inflammation in the liver. Furthermore, saponin extracts promoted the diversity of the fecal bacterial colonies which has a close correlation with lipid metabolism. The results above revealed that T. ananas saponin extracts may be a beneficial natural resource for the prevention of atherosclerosis by modulating lipid metabolism.

Background: Hypercholesterolemic mice lacking factors required for activation of CD4 + T cells are characterized by reduced development of atherosclerosis. Consequently, it has been assumed that atherosclerosis involves loss of tolerance against modified self-antigens generated in response to hypercholesterolemia and that presentation of such antigens on major histocompatibility complex class II (MHCII) leads to activation of proatherogenic Th1 cells. In this study, we wanted to determine the role of antigen presentation on MHCII in atherosclerosis development. Methods: Apolipoprotein E (ApoE −/− ) mice deficient for MHCII (ApoE −/− MHCII −/− ) were used to study the role of MHCII in atherosclerosis development. Results: Compared with ApoE −/− mice, ApoE −/− MHCII −/− mice had reduced levels of CD4 + T cells, immunoglobulin G and M levels, and Th1 and Th2 cytokines in plasma. CD8 + T cells were increased and regulatory T cells were reduced both in spleen and in lesions of ApoE −/− MHCII −/− mice. Decreased plasma levels of inflammatory cytokines in ApoE −/− MHCII −/− mice indicated reduced systemic inflammation. Despite this, ApoE −/− MHCII −/− mice had significantly more atherosclerosis as assessed by en face Oil Red O staining of the aorta (4.7±2.9% versus 1.9±1.3%; P 5 µm 2 versus 4.6±2.8×10 5 µm 2 ; P −/− MHCII −/− mice. Conclusions: Our observations demonstrate that antigen presentation on MHCII has important protective functions in atherosclerosis and that this is primarily the result of activation of regulatory T cells. These findings have implications for understanding the possible risks and benefits of immunosuppressive therapy in patients with cardiovascular disease.

Cardiovascular diseases caused by atherosclerosis have become one of the leading cause of death. The aim of the present study was to explore how oat fiber regulated lipid metabolism and to elucidate its anti-atherosclerotic potential in ApoE−/− mice. Animals were daily fed a high-fat diet without or with 0.8% oat fiber for 18 weeks. Both serum total cholesterol and low-density cholesterol levels decreased in mice received oat fiber administration than those without Oat fiber inhibited hepatic lipid accumulation by downregulating SREBP-1 expression and accelerated clearance of intestinal cholesterol by upregulating SREBP-2 expression. In addition, oat fiber activated cholesterol sensors LXRα, and then strengthened hepatic and intestinal cholesterol efflux by increasing ATP-binding cassette A1 and G1, and decreased intestinal cholesterol absorption by inhibiting Niemann pick C 1 like 1. The findings implicated that oat fiber had an anti-atherosclerotic potential via mechanisms of activating the SREBPs/LXRα pathway and improving lipid metabolism.

Information on the common carotid artery and cerebral microcirculation can be obtained by micro-ultrasound (µUS). The aim of the study described here was to investigate high-fat diet-induced alterations in vascular parameters in ApoE–/– mice. Twenty-two ApoE–/– male mice were examined by µUS and divided into the standard diet (ApoE–/–SD) and high-fat diet (ApoE–/–HF) groups. The µUS examination was repeated after 4 mo (T1). Carotid stiffness, reflection magnitude and reflection index were measured; the amplitudes of the first (W1) and second (W2) local maxima, the local minimum (Wb) and the reflection index (RIWIA = Wb/W1) were assessed with wave intensity analysis. At T1, ApoE–/–HF mice had increased carotid stiffness (1.48 [0.36] vs. 1.88 [0.51]) and reflection magnitude (0.89 [0.07] vs. 0.94 [0.07]) values. Longitudinal comparisons highlighted increases in carotid stiffness for ApoE–/–HF mice (from 1.37 [0.25] to 1.88 [0.51] m/s) but not for ApoE–/–SD mice (from 1.40 [0.62] to 1.48 [0.36] m/s). ApoE–/–HF mice exhibited carotid artery stiffening and increased wave reflections.

We integrate a biocompatible plant virus-based nanotechnology (tobacco mosaic virus, TMV) with S100A9-targeting peptides for its application in imaging and diagnosis of atherosclerosis. S100A9-targeted TMV nanoparticles exhibit remarkable specificity to S100A9 and targeting of atherosclerosis lesions in ApoE(−/−) mice.

Background: Epidemiologic studies indicate that early life arsenic exposures are linked to an increased risk of cardiovascular diseases. Different oxidation and methylation states of arsenic exist in the environment and are formed in vivo via the action of arsenic (+3 oxidation state) methyltransferase (As3MT). Methylated arsenicals are pro-atherogenic postnatally, but pre- and perinatal effects are unclear. This is particularly important because methylated arsenicals are known to cross the placenta. Objectives: We tested the effects of early life exposure to inorganic and methylated arsenicals on atherosclerotic plaque formation and its composition in apolipoprotein E knock-out (apoE−/−) mice and evaluated whether apoE−/− mice lacking As3MT expression were susceptible to this effect. Methods: We exposed apoE−/− or apoE−/−/As3MT−/− mice to 200 ppb inorganic or methylated arsenic in the drinking water from conception to weaning and assessed atherosclerotic plaques in the offspring at 18 wk of age. Mixed regression models were used to estimate the mean difference in each outcome relative to controls, adjusting for sex and including a random effects term to account for within-litter clustering. Results: Early life exposure to inorganic arsenic, and more profoundly methylated arsenicals, resulted in significantly larger plaques in the aortic arch and sinus in both sexes. Lipid levels in these plaques were higher without a substantial difference in macrophage numbers. Smooth muscle cell content was not altered, but collagen content was lower. Importantly, there were sex-specific differences in these observations, where males had higher lipids and lower collagen in the plaque, but females did not. In mice lacking As3MT, arsenic did not alter the plaque size, although the size was highly variable. In addition, control apoE−/−/As3MT−/− mice had significantly larger plaque size compared with control apoE−/−. Conclusion: This study shows that early life exposure to inorganic and methylated arsenicals is pro-atherogenic with sex-specific differences in plaque composition and a potential role for As3MT in mice. https://doi.org/10.1289/EHP8171

BackgroundTo investigate the effect of Ruanmailing oral liquid on atherosclerosis and TGF-β1/SMAD4 signaling pathway in ApoE knockout mice induced by high-fat diet. MethodsForty ApoE-/- mice were randomly divided into 5 groups, and mice fed with standard diets were the control group. ApoE-/- mice high-fat diet induced atherosclerotic phenotype. After grouping and treatment, they were divided into high-fat feeding model group, low-dose and high-dose of Ruanmailing groups (1.75, 4.55 ml/kg/d), Lipitor Group (3.0 mg/kg/d). After 12 weeks of administration, blood was collected from the mice orbit to determine the levels of TC, TG, LDL-C, and HDL-C, and the pathological changes of thoracic aorta atherosclerosis were observed. Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of serum TGF-β1, and RT-PCR and Western Blot were used to detect the expression of SMAD4 and GATA2 in the thoracic aorta of ApoE-/- mice in each group. ResultsCompared with the high-fat model group, the serum lipids level of each administration group were reduced (PPP-/- mice were alleviated, and the high-dose Ruanmailing group had the most significant anti-atherosclerotic effect. ConclusionsRuanmailing oral liquid has an anti-atherosclerotic effect, and its mechanism may be related to the intervention of GATA2 in the TGF-β1/SMAD4 signaling pathway to reduce the differentiation and proliferation of arterial smooth muscle cells.

IntroductionAtherosclerosis has become a worldwide medical burden. Our previous studies have shown that Artemisinin(ART) had the capability to reduce atherosclerosis. Emerging evidence indicates that Long non-coding RNAs(lncRNAs) are engaged in the formation of atherosclerosis. However, whether lncRNAs might participate in the mechanism through which artemisinin mitigates atherosclerosis has not been reported.Material and methodsEight-week-old apolipoprotein E deficient (APOE-/-) mice were divided into two groups, one of which was treated with Artemisinin. Red oil O staining was used to measure the sizes of Atherosclerotic lesion. We conducted deep sequencing to investigate lncRNA profiles in the aorta tissue in high-fat diet fed APOE knockdown mice with and without artemisinin treatment. CeRNA network, Kyoto Encyclopedia of Genes and Genomes(KEGG) and Gene Ontology(GO) analyses were constructed through bioinformatics analysis. RT-PCR was used to validate the differentially expressed lncRNAs.ResultsA total of 102 lncRNAs and 4,630 mRNAs were differentially expressed (pConclusionsThese findings indicated the possible mechanism and therapeutic role of lncRNAs in Artemisinin treatment of atherosclerosis and provided a theoretical basis for the future application of Artemisinin in patients with atherosclerosis.

Objective: Hyperleptinemia, hallmark of obesity, is a putative pathophysiologic trigger for atherosclerosis. We previously reported a stimulatory effect of leptin on TSP-1 (thrombospondin-1) expression, a proatherogenic matricellular protein implicated in atherogenesis. However, a causal role of TSP-1 in leptin-driven atherosclerosis remains unknown. Approach and Results: Seventeen-weeks-old ApoE −/− and TSP-1 −/− /ApoE −/− double knockout mice, on normocholesterolemic diet, were treated with or without murine recombinant leptin (5 µg/g bwt, IP) once daily for 3 weeks. Using aortic root morphometry and en face lesion assay, we found that TSP-1 deletion abrogated leptin-stimulated lipid-filled lesion burden, plaque area, and collagen accumulation in aortic roots of ApoE −/− mice, shown via Oil red O, hematoxylin and eosin, and Masson trichrome staining, respectively. Immunofluorescence microscopy of aortic roots showed that TSP-1 deficiency blocked leptin-induced inflammatory and smooth muscle cell abundance as well as cellular proliferation in ApoE −/− mice. Moreover, these effects were concomitant to changes in VLDL (very low-density lipoprotein)-triglyceride and HDL (high-density lipoprotein)-cholesterol levels. Immunoblotting further revealed reduced vimentin and pCREB (phospho-cyclic AMP response element-binding protein) accompanied with augmented smooth muscle-myosin heavy chain expression in aortic vessels of leptin-treated double knockout versus leptin-treated ApoE −/− ; also confirmed in aortic smooth muscle cells from the mice genotypes, incubated ± leptin in vitro. Finally, TSP-1 deletion impeded plaque burden in leptin-treated ApoE −/− on western diet, independent of plasma lipid alterations. Conclusions: The present study provides evidence for a protective effect of TSP-1 deletion on leptin-stimulated atherogenesis. Our findings suggest a regulatory role of TSP-1 on leptin-induced vascular smooth muscle cell phenotypic transition and inflammatory lesion invasion. Collectively, these results underscore TSP-1 as a potential target of leptin-induced vasculopathy.

Chemokines may promote the formation and instability of atherosclerotic plaque, which is the most common cause of acute coronary syndrome. The aim of this study was to clarify the function of monocyte chemotactic protein-3 (MCP-3) in the stability of atherosclerotic plaque, to determine the role of tissue factor pathway inhibitor (TFPI) on the development and stability of atherosclerotic plaques, and to further elucidate the anti-atherosclerotic mechanism of TFPI with the emphasis on chemokine MCP-3. We constructed an adenovirus-mediated shRNA against mouse MCP-3 (Ad-MCP-3-shRNA) and an adenovirus-containing TFPI (Ad-TFPI), and tranferred them in a model of vulnerable plaque in ApoE-/- mice respectively. Here, we reported that MCP-3-shRNA and TFPI could both reduce the plaque area and decrease the content of lipids and macrophages, on the contrary, the fibrous cap thickness and content of collagen and smooth muscle cells were increased. In addition, the expression of MCP-3 and CC chemokine receptor 2 (CCR2) was decreased by TFPI transfer. These data provide the first

Background & AimBile acids, as hydrophilicity/ hydrophobic molecules and natural ligands of bile acids receptors, are involved in the pathogenesis of liver injury and metabolic disorders. Cholestyramine acts as a bile acid chelating agent and is used to reduce cholesterol in clinic, but its hepatotoxicity and effects on metabolism are still unclear.MethodsApoE-/- mice were continuously administered with low fat diet with or without 2% cholestyramine 12 weeks to determine the hepatotoxicity and metabolic influences of cholestyramine on these mice. Serum transaminases, liver histological features and hepatic inflammatory markers expression were used to assess cholestyramine hepatotoxicity. Body weight, liver weight, serum lipids profile and liver lipid metabolism related genes expression were used to evaluate effects of cholestyramine on metabolism. Differences of endogenous bile acids composition, bile acids metabolism related genes and intestinal flora structure between groups were also analyzed by an ultraperformance liquid chromatography coupled to tandem mass spectrometry system, quantitative real time PCR and 16S rDNA sequencing.ResultsAfter 12 weeks of dietary intervention, although cholestyramine decreased ApoE-/- mice serum cholesterol level, it significantly increased the body and liver weight of these mice, and caused severe liver injury: increased serum transaminases levels, promoted liver vesicular steatosis and inflammatory cells infiltration, and enhanced the expression of lipid synthesis gene: FASN and inflammatory markers genes: TNF-α 、MCP-1、F4/80 and CD68. Moreover, cholestyramine administration caused hydrophobic transformation of bile acids pool and severe gut dysbiosis, which were characterized by decreased hydrophilic bile acids: muricholic acid (MCA) and ursodeoxycholic acid (UDCA), and increased abundance of potential pathogenic bacteria: s_Bacteroide_vulgatus.ConclusionThis study revealed that cholestyramine had the ability to induce severe liver injury and metabolic disorders. These effects might be attributed to inducing hydrophobic transformation of bile acids pool and gut dysbiosis by cholestyramine. Furthermore, our study provided a warning of hepatotoxicity and metabolic disorders for clinical application of cholestyramine, and suggested that cholestyramine was not a good scavenging tool to study the role of bile acids alternations in diseases progression.

Introduction: Lysyl oxidase (LOX) is a copper-dependent amino-oxidase that initiates covalent cross-linking of collagen and elastin, modifies growth factor action and controls gene expression. LOX upregulation is associated with cardiovascular diseases. Because embryonic LOX knockout (KO) mice die perinatally of aortic aneurysms and cardiovascular dysfunction, the studies about LOX function in cardiovascular disease has relied mostly on the use of beta-aminopropionitrile. However, this irreversible inhibitor also inhibits other members of LOX family. Hypothesis: To overcome this limitation, we hypothesized that conditional inactivation of lysyl oxidase would improve vascular function in apoE -/- mice. Methods: We developed, for the first time, a global (LOX f/f CAG-CreERT2 ApoE -/- ) and a smooth muscle cell (SMC) specific (LOX f/f Myh11-CreERT2 ApoE -/- ) LOX conditional knockout mice. Results: LOX inactivation in SMC decreased immature collagen crosslinking in the aorta, carotid pulse wave velocity and blood pressure. It led to a significant reduction in atherosclerotic plaque deposition in the aorta after 16 weeks of high-fat diet (HFD). Global inactivation of LOX, on the other hand, also reduced the systolic blood pressure and prevented vascular stiffness after 4 weeks of HFD. In in-vitro studies, we found that LOX is present in the extracellular space and the nucleus of SMC. Bulk RNA-seq revealed that LOX deletion elevated the expression of SMC alpha-actin, transgelin, and filamin A genes, which are typically present in the contractile phenotype. Conclusion: Our data show that conditional deletion of LOX is atheroprotective and improved vascular and hemodynamic function in ApoE KO mice.

Intraplaque calcification is a prominent feature of advanced atherosclerotic plaque development. Current clinical evidence suggests that the size of calcium deposit may confer different effects on plaque stability [1], [2], [3]. Macro-calcified deposits (CT detected) are thought to confer plaque stability whereas micro-calcification ([18F]NaF PET detected) are thought to be a feature of high-risk ‘vulnerable’ plaques which are prone to rupture. Following on from the emerging role of micro-calcification in high risk plaques within the clinic [4], there is now an urgent need for preclinical atherosclerotic models with this feature to gain mechanistic insights and assess the impact of calcification-targeted therapies. Using a combination of invasive and ex vivo methods, ApoE−/− mice placed on an atherogenic diet have been shown to develop intraplaque calcification [5]. Additionally, [18F]NaF PET/CT has been used to assess the impact of exercise on calcification in ApoE−/− mice on a western diet [6]. In this study, we set out to determine if [18F]NaF PET/CT could be used to non-invasively detect and quantify micro-calficiation in the ApoE−/− high cholesterol diet (HCD) mouse model, and examine the temporal nature of this process.

Background Interferon regulatory factor (IRF) 5 is a transcription factor promoting inflammatory macrophage polarization (M1 type). Given the central role of macrophages in atherosclerotic plaque development we hypothesized that macrophage specific deletion of IRF5 will protect from atherosclerosis. Purpose Investigate whether intrinsic blockade of M1 macrophage polarization ameliorates atherosclerosis Methods Female ApoE−/−LysMCre/wtIRF5flox/floxand ApoE−/−LysMwt/wtIRF5flox/floxmice were fed a high cholesterol diet for 3 months, and atherosclerotic plaque size and compositions as well as inflammatory gene expression were analyzed. Mechanistically, IRF5-dependend bone marrow derived macrophage cytokine profiles were tested under M1 and M2 polarizing conditions. Aortic macrophage chimerism in irradiated ApoE−/− mice reconstituted with a mixture of CD45.1+ ApoE−/− (WT) and CD45.2+ ApoE−/− LysMCre/WtIRF5flox/flox(KO) bone marrow was evaluated to distinguish systemic from intra-plaque effects on monocyte/macrophage kinetics. Results Macrophage-specific IRF5 deficiency blunted LPS/IFNg-induced IL-1β and TNFα gene expression in vitro. In ApoE−/− mice, macrophage-specific IRF5 deficiency did not alter lesion size in the aortic root but significantly reduced macrophage and lipid contents by about 25% while increasing collagen deposition by over 30%. This was accompanied by relative reductions in gene expressions of pro-inflammatory (IL-1β, IL-6, IL-12) and increases in anti-inflammatory (Mertk, TGFβ, CD206) markers in atherosclerotic aortas of ApoE−/−LysMCre/wtIRF5flox/floxmice. When competing with IRF5 deficient cells in mixed irradiation bone marrow chimeras, IRF5 competent macrophages showed an advantage in accumulating in atherosclerotic aortas as disease progressed independent of monocyte recruitment. Conclusion Transcription factor IRF5 promotes a pro-inflammatory response in macrophages leading to vulnerable plaque formation and plaque destabilization, providing genetic evidence for targeting macrophage polarization in atherosclerosis. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DFG

Background Aortic valve disease is the most common valvular heart disease leading to valve replacement. The efficacy of pharmacological therapy for aortic valve disease is limited by the high mechanical stress at the aortic valves impairing the binding rate. We aimed to identify nanoparticle coating with entire platelet membranes to fully mimic their inherent multiple adhesion mechanisms and target the sclerotic aortic valve of apolipoprotein E-deficient (ApoE−/−) mice based on their multiple sites binding capacity under high shear stress. Methods Considering the potent interaction of platelet membrane glycoproteins with components present in sclerotic aortic valves, platelet membrane-coated nanoparticles (PNPs) were synthetized and the binding capacity under high shear stress was evaluated in vitro and in vivo. Results Compared with PNPs bound intensity in the static station, 161%, 59%, and 39% of attached PNPs remained adherent on VWF-, collagen-, and fibrin-coated surfaces under shear stress of 25dyn/cm2 respectively. PNPs demonstrated effectively adhering to von Willebrand factor, collagen and fibrin under shear stresses in vitro. In an aortic valve disease model established in ApoE−/− mice, PNPs group exhibited significant increase of accumulation in the aortic valves compared with PBS and control NP group. PNPs displayed high degrees of proximity or co-localization with vWF, collagen and fibrin, which exhibited good targeting to sclerotic aortic valves by mimicking platelet multiple adhesive mechanisms. Conclusion PNPs could provide a promising platform for the molecular diagnosis and targeting treatment of aortic valve disease. Targeting combination Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): National Natural Science Foundation of China

Background: Atherosclerosis is a major pathogenic factor in cardiovascular diseases during the aging process. Foam cell formation and endothelial dysfunction play a key role in the initiation and development of atherosclerosis, which are affected by lipid disorder and inflammation. Therefore, the drug for inhibition of endothelial dysfunction and the subsequent foam cell formation is a therapeutic approach for atherosclerosis treatment. Total flavone of Astragalus membranaceus (TFA) is extracted from Astragalus membranaceus that has protective effect on cardiovascular disease. However, the effect of TFA on atherosclerosis and the underlying mechanism remains unknown. Methods: In this study, we determined whether TFA could inhibit atherosclerosis; and uncovered the underlying molecular mechanisms. In vivo, apoE deficient mice were treated with TFA contained in high-fat diet for 16 weeks. After treatment, aorta, macrophage and serum samples were collected to determine atherosclerotic lesions, lipid metabolism, and expression of associated genes. Concurrently, we investigated the effect of TFA on monocyte adhesion, foam cell formation, endothelial activation, and macrophage polarization in vitro and in vivo. Results: TFA reduced atherosclerotic plaque size; and enhanced lesion stability by changing the composition of plaque. Moreover, foam cell formation and its accumulation in arterial wall were attenuated by TFA, which might be attributed to improved lipid disorder, reduced inflammation, and decreased monocyte adhesion to endothelial cells. Mechanistically, TFA reduced the expression of scavenger receptor, such as CD36 and SRA; promoted the expression of ATP-binding cassette transporter A1 and G1 (ABCA1/G1). More importantly, TFA reduced the expression of miR-33, a negative regulator of cholesterol efflux as well as positive mediator on inflammation; and concurrently reduced NFkB activity, by which de-repressed ABCA1/G1 activity and inhibited the inflammation. Conclusions: This study demonstrates that TFA can attenuate atherosclerosis via dual inactivation of miR-33 and NFkB signaling pathway and inhibition of scavenger receptors (CD36 and SRA), which suggests that TFA might be a novel therapeutic approach for inhibition of atherosclerosis.

Supplementation of probiotics is a promising gut microbiota-targeted therapeutic method for hyperlipidemia and atherosclerosis. However, the selection of probiotic candidate strains is still empirical. Here, we obtained a human-derived strain, Lactobacillus mucosae A1, which was shown by metagenomic analysis to be promoted by a high-fiber diet and associated with the amelioration of host hyperlipidemia, and validated its effect on treating hyperlipidemia and atherosclerosis as well as changing structure of gut microbiota in ApoE-/- mice on a Western diet. L. mucosae A1 attenuated the severe lipid accumulation in serum, liver and aortic sinus of ApoE-/- mice on a Western diet, while it also reduced the serum lipopolysaccharide-binding protein content of mice, reflecting the improved metabolic endotoxemia. In addition, L. mucosae A1 shifted the gut microbiota structure of ApoE-/- mice on a Western diet, including recovering a few members of gut microbiota enhanced by the Western diet. This study not only suggests the potential of L. mucosae A1 to be a probiotic in the treatment of hyperlipidemia and atherosclerosis, but also highlights the advantage of such function-based rather than taxonomy-based strategies for the selection of candidate strains for the next generation probiotics.

BackgroundThe strategy of decreasing atherosclerotic cardiovascular disorder is imperative to reduce premature death and improve quality of life in patients with diabetes mellitus. The present study was designed to investigate whether the natural flavone acacetin could improve diabetes- accelerated atherosclerotic lesions.MethodsDiabetic model was established in 7-week-old ApoE−/− mice by intraperitoneal injection of STZ (daily 50 mg/kg) for 5 days. Animals of control, control with acacetin treatment, STZ-diabetes, STZ-diabetes with acacetin treatment received acacetin prodrug subcutaneously (20 mg/kg, b.i.d.) or equivolume saline for 12 weeks, and the elasticity of carotid artery and the ability of vascular wall movement were determined with ultrasound and magnetic resonance imaging. Human umbilical vein endothelial cells (HUVECs) were cultured with medium containing 5.5 mM or 33 mM glucose and treated with acacetin or vehicle. Changes of related aortic lesions and signaling molecules were determined by biochemical and molecular approaches in animals and cultured HUVECs.ResultsIt was found that acacetin significantly suppressed atherosclerotic lesions and neointima hyperplasia, improved the elasticity of carotid artery and the ability of vascular wall movement without reducing blood glucose level and reversed the impaired signaling molecules (i.e. SOD, Bcl2, PGC-1α, pAMPK, Sirt3 and Sirt1) in artery tissues in diabetic mice. In cultured HUVECs, high glucose-induced cell viability reduction, ROS over-production, decrease of anti-oxidation, increase of apoptosis, and impairment of mitochondrial function were countered by acacetin (0.3-3 µM) in a concentration-dependent manner. Moreover, acacetin relies on Sirt1 activation by increasing NAMPT and NAD+ followed by Sirt3, pAMPK and PGC-1α activation. Silencing Sirt1 abolished acacetin-induced activation of Sirt3, pAMPK, and PGC-1α.ConclusionsThese results indicate that Sirt1-mediated activation of pAMPK/Sirt3 signals is involved in protective effects of acacetin against atherosclerosis in diabetes by preserving mitochondrial function via reducing mitochondrial apoptosis and ROS production and enhancing its biogenesis, which suggests that acacetin may be a drug candidate for reducing atherosclerotic cardiovascular disorder in patients with diabetes.

Background: A 58-year-old lady was introduced to the clinic because of acute coronary syndrome combined with essential thrombocythemia. After coronary artery bypass grafting. After treating her with aspirin, statins and hydroxyurea (HU), the plaques in her coronary arteries showed improvement dramatically. Here, we aim to investigate that HU might regress atherosclerosis plaques in ApoE−/− mice and the potential mechanism.Methods: Wild-type (C57BL/6, n = 8) and Apolipoprotein E knockout (ApoE-/-, n = 40) mice were used in atherosclerosis model and medication groups. The mice were separated into 7 groups, including the normal control group, the atherosclerosis model group, the dual antiplatelet therapy group (aspirin, and clopidogrel bisulfate), the low-dose and high-dose HU therapy groups [aspirin, clopidogrel bisulfate, and HU (10 or 20 mg/kg/day)], the positive medicine group (aspirin, clopidogrel bisulfate, and atorvastatin calcium), and the combined medicine treatment group [aspirin, clopidogrel bisulfate, atorvastatin calcium, and HU (10 mg/kg/day)]. Fasting serum and aortic vessels were obtained after experiment. The aortic oil red O, Hematoxylin-eosin, and full-length oil red O staining was performed to evaluate the HU’s efficacy of anti- atherosclerosis, and the investigation of HU mechanisms was carried out in HepG2 cells for proprotein convertase subtilisin/kexin type 9 (PCSK9) level.Results: The oil red O and H&E staining results came out that HU therapy with antiplatelet showed an obvious effect in decreasing atherosclerosis plaques and the effect of HU therapy (10 or 20 mg/kg) was stronger than dual antiplatelet therapy plus statin, without liver and kidney toxicity observed. Furthermore, the combined drugs with HU (10 mg/kg/day), statin and antiplatelet nearly eliminated the plaques. One of the possible mechanisms of HU might be related with the inhibition of PCSK9.Conclusions: A discovery based on clinic reveals that HU regressed atherosclerosis plaques in ApoE-/- mice, which provides us a new insight into anti-atherosclerosis drugs strategy. PCSK9 could be one of the possible mechanisms and further mechanisms need to be explored.

Background and Aims Patients with chronic kidney disease (CKD) present a high rate of cardiovascular mortality mainly associated with endothelial dysfunction, which causes more cardiovascular events in presence of atherosclerosis. Atherosclerosis is characterized by a significant increase of low density lipoproteins (LDL), reactive oxygen species (ROS) and inflammation. ROS can oxidize LDL generating oxidized-LDL (oxLDL) that promotes the development of cardiovascular pathologies. The aim of this study was to evaluate whether oxLDL induce endothelial dysfunction analysing the involvement of vascular fibrosis. Method The model used for in vivo studies was the Knockout apolipoprotein E (KO-apoE) mice, which resemble human atherosclerosis and shown high levels of cholesterol (LDL) that can be oxidized to oxLDL. In mice, blood pressure was registered before sacrificed them. After that, we measured different parameters as serum cholesterol levels, vascular function by vascular reactivity in mesenteric arteries and vascular fibrosis in aorta by Sirius Red staining and by the protein expression of fibronectin and collagen-I by immunohistochemistry. In order to investigate the mechanism of action of oxLDL, in vitro studios were performed on human smooth muscle cells (SMC) incubated with oxLDL at different times. Fibrosis was evaluated by the expression of TGF- β and extracellular matrix proteins such as fibronectin and collagen-I by Western blot and by immunofluorescence. ROS production was also measured by fluorescence confocal microscopy, using the CellROX Deep Red probe. Results KO-apoE mice shown higher levels of serum cholesterol and blood pressure than WT animals. Moreover, KO-apoE mice showed endothelial dysfunction since their arteries were less relaxed and more contracted. In addition, these mice presented thickening of vascular wall (SMC layer), more fibrosis showing intense Sirius Red staining and less expression of elastin, all compatible with their vascular dysfunction compared to WT mice. Furthermore, aortas from KO-apoE mice showed a slight increase in fibronectin and collagen-I expression assessed by immunohistochemistry. In vivo studies were confirmed in vitro after treating SMC with oxLDL. oxLDL induced fibrosis in SMC by increasing TGF-β, fibronectin and collagen-I protein expressions evaluated by Western blot and immunofluorescence assays. Treatment with oxLDL also increased ROS production, which seem to be responsible of oxLDL-induced fibrosis in human SMC, as it was blocked in the presence of the antioxidant N-Acetyl-cysteine. Conclusion In summary, these results point to endothelial dysfunction associated to atherosclerosis (oxLDL) could be mediated by an increase in the development of vascular fibrosis where ROS could play an important role. Therefore, the endothelial dysfunction typical of CKD patients could impair with more vascular fibrosis when atherosclerosis is also present.

OBJECTIVES: High consumption of red meat can lead to cardiovascular disease. L-carnitine is rich in red meat can be metabolized by gut microbiota and hepatic enzyme to produce trimethylamine N-oxide (TMAO), which is the risk factor for cardiovascular disease. Garlic is a traditional food, exhibiting a medicinal effect against many diseases as well as garlic essential oil (GEO) possess anti-oxidation, anti-inflammation, and anti-hyperlipidemic effects to prevent cardiovascular disease. The purpose of this study is to investigate the protective effects of GEO on cardiovascular disease as well as explore the mechanism via modulation of gut microbiota composition and ameliorating lipid profiles. METHODS: GEO was extracted by steam distillation and its chemical constituents were analyzed by gas chromatography. The eight-week-old female apoE(−/−) mice were randomly divided into 5 groups: (1) chow diet, (2) 1.3% carnitine in water-fed group (negative control), (3) 1.3% carnitine + GEO (25 mg/kg bw), (4) 1.3% carnitine + GEO (50 mg/kg bw), and (5) 1.3% carnitine + 1% 3,3-dimethyl-1-butanol in water-fed (positive control). The mice were gavaged with GEO or soybean oil daily after 15 weeks the mice were sacrificed. The whole aorta was collected for observing the aortic plaque formation via oil red staining. The serum cholesterol, triglyceride, HDL, AST, and ALT were measured by an automatic blood analyzer. The bacterial metabolite trimethylamine (TMA) and TMAO were examined by using LC-MS. Illumina Miseq platform was used for 16S rDNA sequencing to analyze the feces microbiota composition. RESULTS: GEO significantly reduced the atherosclerotic lesion area in aorta compared to the carnitine-fed group (P

Background Atherosclerosis (AS), which characterized with the accumulation of lipids on the vessel wall, is the pathological basis of many cardiovascular diseases (CVD) and seriously threatens human health. Resveratrol (RES) has been reported to be benefit for AS treatment. This research aimed to observe the effects of RES on AS induced by high-fat diet (HFD) and LPS in ApoE -/- mice and investigate the underlying mechanism. Methods ApoE -/- mice were fed with HFD companied with LPS to induce AS and RES was administrated for 20 weeks. Splenic CD4 + T cells were cultured and treated with anti-CD3/CD28 together with LPS, and RES was added. Serum lipids and the atherosclerotic areas of aortas were detected. The activation of CD4 + T cells were investigated both in vivo and in vitro and the expression of DNA methyltransferases (Dnmt) in CD4 + T cells were measured. Results In vivo, administration of RES prevented HFD and LPS induced dysfunction of serum lipids including TC (total cholesterol), TG (triglyceride), LDL-C (low density lipoprotein cholesterol) and HDL-C (high density lipoprotein cholesterol), ameliorated the thickened coronary artery wall and decreased the areas of atherosclerotic lesion on aortas. Besides, RES decreased the number of CD4 + T cells in peripheral blood, decreased the expression of CD25 and CD44, but not affected the expression of L-selectin (CD62L). In vitro, RES decreased the expression of Ki67, CD25 and CD44 in CD4 + T cells. Moreover, RES increased the secretion of IL-2, IL-10 and TGF-β1, decreased IL-6. In addition, RES decreased both the mRNA and protein level of Dnmt1 and Dnmt3b in CD4 + T cells. Conclusion These results indicated that RES ameliorated AS induced by HFD companied with LPS in ApoE -/- mice and inhibited the proliferation and activation of CD4 + T cells by regulating the expression of Dnmt1 and Dnmt3b.

Atherosclerosis (AS) is a complex, chronic inflammatory disease that is characterized by plaque buildup within arterial vessel walls. Preclinical trials have suggested that vorinostat, a pan-histone deacetylase inhibitor (HDACi), reduces vascular inflammation and AS, but the underlying protective mechanism has not been fully elucidated. The present study aimed to identify altered gene expression profiles in aortic tissues from ApoE

Atherosclerosis is the underlying basis for most cardiovascular diseases. It is a chronic inflammation affecting the arterial intima and is promoted by hypercholesterolemia. Cells of both the innate and adaptive immune systems contribute to this inflammation with macrophages and T cells being the most abundant immune cells in the atherosclerotic plaques. In this review, we discuss the studies that examined the role of T cells and T cell subsets in Apoe−/− and Ldlr−/− murine models of atherosclerosis. While there is a general consensus that Th1 cells are pro-atherogenic and regulatory T cells are atheroprotective, the role of other subsets is more ambiguous. In addition, the results in the two models of atherosclerosis do not always yield similar results. Additional studies in the two murine models using cell specific gene manipulations are needed.

Exercise reduces the risk of breast cancer development and improves survival in breast cancer patients. However, the underlying mechanisms of this protective effect remain to be fully elucidated, and it is unclear whether exercise can attenuate the protumor effects of obesity and related hyperlipidemia on breast cancer growth and development. We hypothesized that exercise attenuates the negative effect of hyperlipidemia through normalization of the tumor microenvironment and improved T cell infiltrate. Hyperlipidemic ApoE−/− mice with orthotopic EO771 breast tumors were randomly assigned to one of two voluntary running groups or sedentary controls, and muscular cytochrome c oxidase subunit IV (COX-IV) expression was used as a biomarker for the level of exercise. Tumors from mice with high muscular COX-IV expression took significantly longer to reach 100 mm3 ( P = 0.008), but showed no difference in growth rate once the tumor was established. Wheel running appeared to reduce internal metastases, but did not affect T cell infiltrate or the proportion of regulatory and cytotoxic T cells within the tumor. Serum levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased by tumor burden ( P = 0.02) and correlated with spleen weight ( P < 0.0001, R = 0.65). Furthermore, tumor hypoxia was significantly decreased in mice with high muscular COX-IV expression ( P = 0.01). Taken together, these results indicate that wheel running can slow the establishment of primary and secondary EO771 breast tumors and induce beneficial changes in the breast tumor microenvironment in ApoE−/− mice. NEW & NOTEWORTHY In this first study to investigate the effect of exercise on tumor behavior in a hyperlipidemic model, we hypothesized that wheel running would counteract the protumorigenic environment generated by hyperlipidemia. Wheel running slowed establishment of primary and secondary tumors and reduced tumor hypoxia but did not affect exponential tumor growth in ApoE−/− mice. Overall, voluntary wheel running induced favorable microenvironmental changes in breast tumors.

Cholesterol metabolism becomes imbalanced during the formation of macrophage-derived foam cells. Pre-B-cell colony-enhancing factor (PBEF) has recently been found to affect lipid deposition and inflammation in atherosclerosis. Here, we aimed to study the effects and molecular mechanism of Polydatin on atherosclerosis in ApoE-knockout (ApoE[Formula: see text]) mice. Thirty ApoE[Formula: see text] mice were fed a high-fat diet (HFD) for 12 weeks, and then treated with Polydatin for another 12 weeks. Whole aortas and cryosections were stained with oil red O. Blood lipid, PBEF and cytokine levels were measured by ELISA. The mRNAs of cholesterol metabolism-related genes were determined by qRT-PCR and protein levels by Western blotting. Cell cholesterol content and viability were determined in macrophages and RAW 264.7 cells. PBEF siRNA was used to study the effect of Polydatin on cholesterol metabolism in macrophages incubated with ox-LDL. Polydatin lowered blood lipids and decreased atherosclerotic lesions in ApoE[Formula: see text] mice. The expression of cytokines and the mRNA of cholesterol metabolism-related genes were markedly regulated by Polydatin. Meanwhile, PBEF mRNA and protein were both greatly down-regulated by Polydatin. In vitro, Polydatin protected RAW 264.7 cells treated by ox-LDL and inhibited cholesterol uptake by macrophages. The PBEF siRNA result indicates that Polydatin can modulate cholesterol metabolism in macrophages, partly through down-regulation of PBEF. In conclusion, Polydatin relieves atherosclerosis injury in ApoE[Formula: see text] mice, mainly through down-regulation of PBEF and inhibition of PBEF-inducing cholesterol deposits in macrophages.

Atherosclerosis is the development of lipid-laden plaques in arteries and is nowadays considered as an inflammatory disease. It has been shown that high doses of ionizing radiation, as used in radiotherapy, can increase the risk of development or progression of atherosclerosis. To elucidate the effects of radiation on atherosclerosis, we propose a mathematical model to describe radiation-promoted plaque development. This model distinguishes itself from other models by combining plaque initiation and plaque growth, and by incorporating information from biological experiments. It is based on two consecutive processes: a probabilistic dose-dependent plaque initiation process, followed by deterministic plaque growth. As a proof of principle, experimental plaque size data from carotid arteries from irradiated ApoE\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{{-/-}}$$\end{document}-/- mice was used to illustrate how this model can provide insight into the underlying biological processes. This analysis supports the promoting role for radiation in plaque initiation, but the model can easily be extended to include dose-related effects on plaque growth if available experimental data would point in that direction. Moreover, the model could assist in designing future biological experiments on this research topic. Additional biological data such as plaque size data from chronically-irradiated mice or experimental data sets with a larger variety in biological parameters can help to further unravel the influence of radiation on plaque development. To the authors’ knowledge, this is the first biophysical model that combines probabilistic and mechanistic modeling which uses experimental data to investigate the influence of radiation on plaque development. Electronic supplementary material The online version of this article (doi:10.1007/s00411-017-0709-2) contains supplementary material, which is available to authorized users.